WRN promotes bone development and growth by unwinding SHOX-G-quadruplexes via its helicase activity in Werner Syndrome

Werner Syndrome (WS) is an autosomal recessive disorder characterized by premature aging due to mutations of the WRN gene. A classical sign in WS patients is short stature, but the underlying mechanisms are not well understood. Here we report that WRN is indispensable for chondrogenesis, which is the engine driving the elongation of bones and determines height. Zebrafish lacking wrn exhibit impairment of bone growth and have shorter body stature. We pinpoint the function of WRN to its helicase domain. We identify short-stature homeobox (SHOX) as a crucial and direct target of WRN and find that the WRN helicase core regulates the transcriptional expression of SHOX via unwinding G-quadruplexes. Consistent with this, shox−/− zebrafish exhibit impaired bone growth, while genetic overexpression of SHOX or shox expression rescues the bone developmental deficiency induced in WRN/wrn-null mutants both in vitro and in vivo. Collectively, we have identified a previously unknown function of WRN in regulating bone development and growth through the transcriptional regulation of SHOX via the WRN helicase domain, thus illuminating a possible approach for new therapeutic strategies.

1. This zebrafish mutant appears to be a good WS model, and the study focuses exclusively on the skeletal phenotype in order to unveil the underlying molecular mechanism(s). Do the authors have any knowledge if this zebrafish wrn-/-mutant features any other phenotypes known to be associated to WS? (e.g. premature replication senescence; increased chromosome instability; increased apoptosis; skin defects, like thinning). These would be valuable information since the mouse homolog mouse mutant does not phenocopy so effectively the WS. 2. Figure 2, Panels o-v showing the extent of cartilage formation and proliferative status between WT and Wrn-/-mutant reveal less cartilage differentiation as well less proliferative activity in the mutant. How the authors comment this observation? Proliferation and differentiation are usually two mutually exclusive events during development. 3. Figure 3 and Sup Fig.2, show cartilage formation, under the material and methods section the authors when describe the chondrogenic procedure state that chondrocyte differentiation from hESCs was performed following a previously published protocol (32), referring to Bianco P et al. Skeletal stem cells. Development 142 1023-1027 (2015). This paper is a review and importantly, Bianco and Robey in the context of chondrogenic differentiation assay procedure to follow state that "Cartilage formation in micro-mass or pellet cultures is more reliable, as it rests on ultimate histological proof of genuine cartilage". The authors have performed their chondrogenic assay on cell-monolayer instead….. 4. Apoptosis is known to be a crucial step during endochondral ossification, additionally misregulation of genes involved in apoptosis has been reported in mice lacking a functional Wrn protein and Wrn knockdown inhibits proliferation while promoting apoptosis. However, the authors have completely ignored this aspect. Therefore, investigation on apoptosis would be desirable in order to rule out that potential cell competent to specify towards the chrondrogenic lineage are negatively selected by apoptosis. 5. Supp. Figure 4, Panel g, illustrates FISH analysis of shox expression in WT zebrafish and wrn-/mutant zebrafish 7 dpf. It would be of interest to analyze the expression at other time points, such as dpf 3 and dpf 14and in parallel to the expression of wrn. This profiling would provide better and complete information regarding the co-expression of these two genes during chondrogenesis. 6. Figure 7, panels k-m and u-w show FISH analysis for Col2a1a and col10a1a expression combined with BrdU staining. The authors comment Shox1 overexpression in wrn-/-zebrafish "significantly enhanced " the expression of these genes as well the proliferation activity. It is hard to believe that a staining can be quantified as significant….. I would suggest a qRT in order to establish if the outcome is significant. Moreover, in these panels it is hard to appreciate Sox9 and Col 10a1a staining, whereas it is possible for Col2a1a.
Reviewer #3 (Remarks to the Author): Werner syndrome (WS) is a rare form of accelerated ageing disease with clinical characteristics resembling those of normal ageing. WS is caused by multiple mutations in the gene encoding the Werner DNA helicase (WRN), but the underlying mechanisms driving short stature in WS patients remains elusive. In this paper, Tian et al., investigated the role of WRN gene in regulating of chondrogenesis and the underlying mechanisms using zebrafish as an in vivo model. The authors found that WRN deficiency results in the inhibition of bone growth and short stature. Moreover, the authors, using in vitro cultured human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs), demonstrated that WRN deficiency impairs cartilage development. The authors also performed RNA-seq and ChIP-seq and showed that the SHOX (short-stature homeobox) gene is a direct target of WRN. WRN regulated SHOX expression by facilitating gene transcription. In addition, the authors demonstrated that manipulating the expression of SHOX is sufficient to mimic or rescue chondrogenesis and bone formation in WT or WRN knockdown models. The authors should be commended on their well-conducted study and engaging manuscript, which contains an impressive amount of data that are interpreted to support some interesting conclusions. I have the following points for the authors to address:

Major comments
It has been recognized that DNA damage, cellular senescence, telomere attrition, and impaired autophagy/mitophagy resulting from mutations in WRN explain the majority of the clinical features of WS. It is somewhat disappointing that these hallmark changes were not evaluated in the testing model systems. Particularly, from the BrdU labeling ( Figure 2) and the RNA-seq data ( Figure 5), cell proliferation/cell growth-associate genes are among the most significantly downregulated pathways in the WRN deficiency cells (vs. WT cells), suggesting that cellular senescence is likely involved in WRN deficiency-caused bone development and growth defects. A previous report, which is closely related to the current study, showed that stem/progenitor cells in metaphysis of long bone during rapid growth period are highly proliferative but undergo progressive cell senescence during late puberty when bone growth slows down (Nat. Commun. 2017;8:1312), indicating that cellular senescence is a key mechanism to control bone growth. Therefore, it would be interesting for the authors to assess whether loss of WRN causes accumulation of senescent cells in the zebrafish and the in vitro stem cells culture model systems. It is also interesting to test whether overexpressing SHOX prevented the senescence phenotype.
Minor points 1. The authors should give an overview in the Introduction section on the current understanding of the cellular and molecular mechanisms that result in the accelerated aging of WS patients.
genetic mode of action in the context of the multiplicity of transcription factors coming into play. Summary: Skeletal dysplasia and short stature are clinical phenotypes of considerable complexity with the large number of genes and spectrum of clinical disorders. SHOX is a key regulator of chondrocyte biology, as reflected by the multiple number of target genes that it regulates. While zebrafish serve as a useful model for whole body characterization of genes regulating height and skeletal components, the pathways are quite complex. Moreover, relating skeletal phenotypes of zebrafish to human and tissuespecific gene regulation is formidable. In the current work, the authors have investigated the hypothesis that the WRN helicase-nuclease implicated in the premature aging Werner syndrome regulates expression of SHOX via its G4 resolvase activity on SHOX-promoter associated Gquadruplexes.
The authors document shortened body length and bone abnormalities in wrn-/-zebrafish. From these studies they transition to human embryonic stem cells and human mesenchymal stem cells to examine the role of WRN in chondrogenesis at the cellular level. However, the direct connection of their observations remain elusive to the zebrafish model findings. To their credit, the authors assess if human WRN mRNAs injected into wrn-/-mutant zebrafish at the one-cell stage on regulation of expression of key genes in the chondrogenesis pathway. By this analysis, they find that SHOX expression is regulated by WRN during chondrocyte differentiation, and this is largely dependent on WRN helicase activity. SHOX expression on stature was further examined by microinjection of human SHOX mRNA into zebrafish embryos at the one-cell stage and embryo length was correlated with bone growth/development expression. Finally, the authors determine that WRN unwinds promoterassociated G4, thereby regulating its expression.
Critical Comments: SHOX signaling is notably complex, and there is still only limited understanding of the pleiotropic effects of SHOX deficiency in humans. Therefore, the utilization of model genetic organisms with conserved genes and translational value is appreciated. Zebrafish is one such model for studying chondrogenesis, but has its limitations that are apparent at the organismal and tissue levels. A useful tool in the current study might have been G-quadruplex ligands to modulate SHOX promoter function and interrogate WRN involvement. This experimental approach might have been applied in the in vivo model. Specificity of WRN's effect was not addressed. One wonders if the sequence-related BLM helicase which has also been reported to regulate gene expression via its action on G4 promoters would affect the observed phenotypes via SHOX. In my mind, it remains unclear if the effect of WRN is specific to regulation of SHOX or if other gene regulatory factors are implicated in genetic determination of height and bone homeostasis are involved, that are also directly regulated by WRN or indirectly. While the study suggests WRN is involved, a direct causal relational and mechanism is not established. Assessment of WRN connected SHOX gene regulation on other tissues of zebrafish could be performed to provide a better impression of the broadness of the gene regulatory network. As shown by the authors, 380 genes were found to be regulated by WRN in chondrocyte homeostasis, 116 up-regulated and 264 down-regulated. Given the large number, it may be that multiple genes coregulated with SHOX or even directly regulated by SHOX are also implicated, as suggested. It would have been informative and supportive of their model if the authors had examined in greater detail the mechanism whereby some of the top candidate involved in chondrogenesis, as evidenced by previous studies, are affected and if their regulation is relevant to the phenotypes of wrn-/-zebrafish model in this work. This would apply to SOX9, SOX5, SOX6 (The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis -PubMed (nih.gov)). The gene regulatory networks of chondrocytes are quite complex, and super enhancers as well as hostone modifications and chromatin accessibility come into play. It has been proposed that multiple transcription factors are involved in chrondrocyte biology. While the evidence from the current study suggests that WRN regulation of SHOX is among these, it is difficult to comprehend the molecular-Based on their results, the authors suggest that WRN controls bone growth and development via its regulation of SHOX. Are there other phenotypes beyond bone-related affected by WRN status and are these also mediated by SHOX? The complexity of WRN's genetic linkage to range of clinical features of accelerated aging. This is compounded by the extensive data that at least in vitro (biochemical), cellbased models, and genetic models, WRN plays roles in transcriptional regulation, DNA repair, replication stress response, genomic stability. So is this an oversimplification that WRN's involvement in controlling bone metabolism in zebrafish is mediated by its effect on SHOX, and there is no consideration of its pan-wide functions in multiple cell types and tissues?
Another layer of potential complexity is added with the idea that WRN's key molecular activity of G4 resolution in the promoter of SHOX underlies the growth/bone phenotypes may be overly simplified. WRN, along with other G4-resolving helicases (e.g., BLM) is thought to play a role in regulation of expression of many genes with G4-laden promoters, not to mention its effect on G4 in other chromosomal regions (e.g., telomeres). I'm not sure all the phenotypes related to chrondrocyte biology hinge on WRN-catalyzed G4 resolution of SHOX promoter DNA sequence elements. This seems to be the driving conclusion of the paper, and I am not sure that other avenues have been explored which potentially contribute to the observations made for wrn-/-zebrafish.
A recently published study by Shin et al., DNA Repair (2021) Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes; Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes -PubMed (nih.gov) reported that wrn-/-zebrafish. Like mre11-/-zebrafish and certain other DNA repair mutants displayed reduced growth and development during the juvenile stage. Interestingly, both mre11acu56/cu56 and wrncu64/cu64 failed to survive to 60 dpf. The similar phenotype raises the question if the genetic defect is attributed to unusual G4 metabolism or more generically a DNA repair defect such as double-strand break repair in which both WRN and MRE11 are implicated. It would have been of interest if the authors of the current study had determined what phenotypes and at what developmental stage were observed in wrn-/-zebrafish that had been exposed to a G4-binding ligand. If the temporal appearance or severity of the observed phenotypes were G4-inducible, then this would support their model. Overall Critique: The study of Tian et al. invokes a model in which zebrafish WRN has importance for bone development and growth, a finding consistent with another recently published study in DNA repair. The apparent advance here is that short stature homobox gene SHOX expression is regulated by WRN, which may be a causative factor for wrn-/-related phenotypes; however, further experimental studies might have more strongly supported this conclusion. Furthermore, the authors of the current study propose that WRN regulates SHOX gene expression via resolution of promoter G-quadruplexes of the SHOX gene; however, it is unclear how specific this function of WRN truly is, given that there are a number of G4resolving helicases in humans. Extrapolation of the findings from zebrafish to human remains uncertain and likely would be more complex in the higher order vertebrate. It remains to be fully understood if the dysregulation of SHOX due to loss of WRN G4 resolvase activity underlies the short stature in humans with WS, or if the molecular-genetic basis is more complex.

Dear Editors and Reviewers,
Thank you very much for your comments concerning our manuscript entitled "NCOMMS-21-39263 WRN promotes bone development and growth by unwinding SHOX-G-quadruplexes via its helicase activity in Werner Syndrome". Your comments, suggestions, and requirements for changes in the manuscript have been very valuable and helpful for revising and improving the manuscript. We have carefully studied all comments and have made corrections which we hope answers the requirements set during the revision. Revised parts of the manuscript are marked in red text. Our point-to-point responses to the reviewers' comments are shown below.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): This work by Tian et al., focuses on Werner syndrome (WS), a monogenic autosomal recessive disorder caused by mutations of WRN gene. Patients affected by WS are characterized by short stature alongside to other phenotypes. Using a zebrafish wrn-/-mutant as a model for WS herein, the authors unveil that wrn is a regulator of bone development and growth through transcriptional regulation of Shox gene. Moreover, they provide evidence that Wrn protein triggers this function by unwinding Shox-g -quadruplex via its helicase domain.
We sincerely appreciate reviewer 1's all the valuable comments which have greatly improved our manuscript. Comments: 1. This zebrafish mutant appears to be a good WS model, and the study focuses exclusively on the skeletal phenotype in order to unveil the underlying molecular mechanism(s). Do the authors have any knowledge if this zebrafish wrn-/-mutant features any other phenotypes known to be associated to WS? (e.g. premature replication senescence; increased chromosome instability; increased apoptosis; skin defects, like thinning). These would be valuable information since the mouse homolog mouse mutant does not phenocopy so effectively the WS.
Response: Thank you for the comments on our wrn -/zebrafish model. As pointed out by the reviewer, the WS zebrafish model is suitable for evaluating skeletal phenotypes related to short stature. In addition to this feature, wrn -/zebrafish show a shorter lifespan compared to wildtype zebrafish, as demonstrated previously (1). They also showed increased cellular senescence and apoptosis, which was examined in a chondrocyte context, and fat loss in the abdomen of adult zebrafish.  Figure 2, Panels o-v showing the extent of cartilage formation and proliferative status between WT and Wrn-/-mutant reveal less cartilage differentiation as well less proliferative activity in the mutant. How the authors comment this observation? Proliferation and differentiation are usually two mutually exclusive events during development.

Response:
We appreciate your valuable comments. Previous studies of WS showed that loss of WRN causes DNA damage (2) that may inhibit differentiation (3, 4) and proliferation (5). Zhang et al detected increased ROS levels, enhanced γH2AX expression levels, and skeletal growth retardation in parathyroid hormone-related peptide (PTHrP) knock-in mice (3). Additionally, Li et al observed that increased apoptosis caused bone loss and inhibited osteoblast differentiation in Atg7 mice (4). Based on these previous studies, we examined the expression level of γH2AX, a DNA damage marker. As shown in Fig. a below (summarized in Supplementary Fig. 2b in the revised manuscript) shown below, the expression level of γH2AX was increased in wrn -/zebrafish compared with that in the wildtype, indicating that loss of wrn led to DNA damage. Additionally, we evaluated apoptosis in the vertebrate regions by detecting in situ apoptosis (Invitrogen, C10618). As shown in Fig. b below (summarized in Supplementary Fig. 2a in the revised manuscript), more apoptosis signals were found in wrn -/zebrafish compared with that in the wildtype, which agreed with our previous results that less proliferative cells were observed in wrn -/zebrafish (summarized in Fig. 2p and r in the revised manuscript). Furthermore, it has been previously reported that DNA damage drives bone aging and inhibits bone differentiation, indicating that senescence plays a critical role in bone differentiation (5). Thus, we measured the expression of senescence markers, namely, p53 and p16, using FISH analysis and observed the up-regulation of p53 and p16 in wrn -/zebrafish ( Fig. c shown below, summarized in Fig. 8k-m in the revised manuscript). We also detected more senescent chondrocytes in both sh-WRN hESCs and sh-WRN hMSC compared with those in the CTR groups using flow cytometry analysis (Fig. e, f, i, and j shown below, summarized in Fig. 8e, f, i, and j), indicating that loss of WRN/wrn could lead to chondrocyte senescence. More importantly, overexpression of SHOX/shox (name as the rescue group) in cells and zebrafish promoted proliferation (data please refer to Fig. 7l, n, p, s, u, and w in the revised manuscript) and prevented the senescence phenotype ( Fig. g, k, l, and m shown below, summarized in Supplementary Fig. 8g, k, l, and m) compared with that in sh-WRN hESCs and sh-WRN hMSCs. Based on these results, WRN/wrn loss-induced apoptosis and senescence is one possible explanation for why both differentiation and proliferation were inhibited. We also added the related contents in the manuscript:  (2015). This paper is a review and importantly, Bianco and Robey in the context of chondrogenic differentiation assay procedure to follow state that "Cartilage formation in micro-mass or pellet cultures is more reliable, as it rests on ultimate histological proof of genuine cartilage". The authors have performed their chondrogenic assay on cell-monolayer instead…..
Response: Thank you for the comment on the reference and protocol. We understand the reviewer's concern regarding the cell-monolayer method, and we apologize for the error in the reference. We followed the protocol described in the article, "Directed Differentiation of Human Embryonic Stem Cells Towards Chondrocytes" (Rachel AO et al. Nature Biotechnology, 2010) (reference number is 35 in the revised manuscript, previously reference number is 52). The advantage of this model (Fig.  a shown below, summarized in Fig.3a in the revised manuscript) is that it fully recapitulates the whole chondrogenic development process from the pluripotent stage to the mesoderm stage; and chondrocyte stage. Pluripotent hESCs (stage 1, day 0) were directed towards a primitive streakmesendoderm population (stage 2, day 4), after which differentiation proceeded to a mesoderm population (stage 3, day 9), and finally towards chondrocytes (stage 4, day 14), which is useful for examining the complete process of chondrocyte differentiation in the context of WS. We also used human mesenchymal stem cells as our second cell model (Fig. b shown below, summarized in Supplementary Fig.3a in the revised manuscript); chondrogenesis in the hMSC model was highly similar to that in the hESC model from stages 3 to 4. We performed micromass cell culture for chondrogenic differentiation of hMSCs. The micromass cell culture provides a three-dimensional environment that allows the cells to grow in aggregates, which is suitable for generating a high cell density and cell-cell interactions to better induce chondrogenesis (6)  (2018) 4. Apoptosis is known to be a crucial step during endochondral ossification, additionally misregulation of genes involved in apoptosis has been reported in mice lacking a functional Wrn protein and Wrn knockdown inhibits proliferation while promoting apoptosis. However, the authors have completely ignored this aspect. Therefore, investigation on apoptosis would be desirable in order to rule out that potential cell competent to specify towards the chrondrogenic lineage are negatively selected by apoptosis.
Response: Thank you for pointing out the essential link between the loss of WRN and the induction of apoptosis and inhibition of proliferation. With regards to bone development, the apoptosis is thought to play a critical role during skeletal development (7). Therefore, we evaluated apoptosis in both cells and zebrafish at different bone developmental stages to exclude the possibility that chondrocytes are negatively selected by apoptosis. As shown by poly-caspases and PI staining, we found that many apoptotic chondrocytes at an early stage both in shWRN1# hESCs and shWRN1# hMSCs (Fig. a-b shown below, summarized in Supplementary Fig. 4a-b). We noted the accumulation of apoptotic cells began appearing at stage 2, which is the premesoderm stage in the hESC model, with more apoptotic chondrocytes accumulated at stage 4 ( Fig. a shown below, summarized in Supplementary Fig. 4a). Similarly, the number of apoptotic chondrocytes increased during bone development in the hMSC model (Fig. b shown below, summarized in Supplementary  Fig. 4b in the revised manuscript). In agreement with the staining results, the apoptosis markers BCL2 and CASPASE 8 showed enhanced expression in shWRN1# hESCs and shWRN1# hMSCs compared to CTR hESCs or hMSCs (Fig. c-f shown below, summarized in Supplementary Fig. 4cf in the revised manuscript). Moreover, a larger number of apoptosis signals were found in wrn -/zebrafish from 3 dpf to 14 dpf compared with that in the wildtype in the vertebrate regions ( Fig. g shown below, summarized in Supplementary Fig.2a in the revised manuscript). Collectively, these results suggest that loss of WRN leads to early apoptosis and exclude the possibility that chondrocytes are negatively selected by apoptosis. We also added the related contents in the manuscript: Page 8 from line 233 to line 237, page 11 from line 342 to line 345 in results section.  Figure 4, Panel g, illustrates FISH analysis of shox expression in WT zebrafish and wrn-/mutant zebrafish 7 dpf. It would be of interest to analyze the expression at other time points, such as dpf 3 and dpf 14and in parallel to the expression of wrn. This profiling would provide better and complete information regarding the co-expression of these two genes during chondrogenesis.
Response: Thank you for the suggestions. We have added more time points for shox expression in both the wildtype and wrn-/-zebrafish, as suggested. As shown below (summarized in Supplementary Fig.7h-j), wrn, shox and BrdU signals accumulated in wildtype zebrafish from 3 to 14 dpf according to FISH analysis; this accumulation was not observed in wrn -/zebrafish, indicating that wrn and shox were co-expressed during chondrogenesis. We also added the related contents in the manuscript: Page 17 from line 456 to line 459.
6. Figure 7, panels k-m and u-w show FISH analysis for Col2a1a and col10a1a expression combined with BrdU staining. The authors comment Shox1 overexpression in wrn-/-zebrafish "significantly enhanced " the expression of these genes as well the proliferation activity. It is hard to believe that a staining can be quantified as significant….. I would suggest a qRT in order to establish if the outcome is significant. Moreover, in these panels it is hard to appreciate Sox9 and Col 10a1a staining, whereas it is possible for Col2a1a.
Response: Thank you for the comments. We have added the qRT-PCR results. These results are shown below and summarized in Fig.7k and r. These results are in agreement with our FISH analysis in Fig. 7, showing that the loss of wrn led to the decreased expression of sox9a, col2a1a, and col10a1a. Moreover, overexpression of shox promoted these genes expression in wrn -/zebrafish. In the meantime, we have corrected our description in the manuscript: Page 22 from line 560 to line 564.
Reviewer #2 (Remarks to the Author): Werner syndrome (WS) is a rare form of accelerated ageing disease with clinical characteristics resembling those of normal ageing. WS is caused by multiple mutations in the gene encoding the Werner DNA helicase (WRN), but the underlying mechanisms driving short stature in WS patients remains elusive. In this paper, Tian et al., investigated the role of WRN gene in regulating of chondrogenesis and the underlying mechanisms using zebrafish as an in vivo model. The authors found that WRN deficiency results in the inhibition of bone growth and short stature. Moreover, the authors, using in vitro cultured human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs), demonstrated that WRN deficiency impairs cartilage development. The authors also performed RNA-seq and ChIP-seq and showed that the SHOX (short-stature homeobox) gene is a direct target of WRN. WRN regulated SHOX expression by facilitating gene transcription. In addition, the authors demonstrated that manipulating the expression of SHOX is sufficient to mimic or rescue chondrogenesis and bone formation in WT or WRN knockdown models. The authors should be commended on their wellconducted study and engaging manuscript, which contains an impressive amount of data that are interpreted to support some interesting conclusions. I have the following points for the authors to address: We are grateful to reviewer 2 for his/her effort in reviewing our manuscript and gave us all the helpful comments.

Major comments
It has been recognized that DNA damage, cellular senescence, telomere attrition, and impaired autophagy/mitophagy resulting from mutations in WRN explain the majority of the clinical features of WS. It is somewhat disappointing that these hallmark changes were not evaluated in the testing model systems. Particularly, from the BrdU labeling ( Figure 2) and the RNA-seq data ( Figure 5), cell proliferation/cell growth-associate genes are among the most significantly downregulated pathways in the WRN deficiency cells (vs. WT cells), suggesting that cellular senescence is likely involved in WRN deficiency-caused bone development and growth defects. A previous report, which is closely related to the current study, showed that stem/progenitor cells in metaphysis of long bone during rapid growth period are highly proliferative but undergo progressive cell senescence during late puberty when bone growth slows down (Nat. Commun. 2017; 8:1312), indicating that cellular senescence is a key mechanism to control bone growth. Therefore, it would be interesting for the authors to assess whether loss of WRN causes accumulation of senescent cells in the zebrafish and the in vitro stem cells culture model systems. It is also interesting to test whether overexpressing SHOX prevented the senescence phenotype.

Response:
We appreciate the valuable comments. Depletion of WRN has been reported to cause accumulation of senescence-associated-β -galactosidase (SA-β -gal) positive hMSCs (8). To determine whether the loss of WRN lead to the accumulation of senescent chondrocytes, we performed senescence assays both in vitro and in vivo. We first examined cellular senescence using senescence flow cytometry analysis (Invitrogen, C10840) by labeling SA-β-gal positive cells. Gating strategy is shown below in Fig. a ( Fig. 8e). Flow cytometry analysis revealed an increased number of SA-βgal-positive cells (P3), with 29.6% senescent cells detected in shWRN1# hESCs and 30.1% in shWRN1# hMSCs; only 0.2% of hMSCs were senescent in the control groups and 0.0% in control hESCs (Fig. b, c, f, and g shown below, summarized in Fig. 8b, c, f, and g). After overexpression of SHOX in shWRN1# hESCs and shWRN1# hMSCs, the number of senescent cells decreased to 13.5% and 14.1%, respectively ( Fig. d and h shown below, summarized in Fig. 8d and h). We also evaluated the expression of two senescence markers, P53 and P16, both in stem cells and zebrafish models. The mRNA expression of P53 and P16 increased in shWRN1# hESCs and shWRN1# hMSCs compared with that in CTR cells; overexpression of SHOX decreased the expression of these two genes ( Fig.k-n, summarized in Fig. 8k-n). Similar results were observed in the zebrafish model at different development stages (Fig. o-q, summarized in Fig. 8o-q). Together, these results indicate that loss of WRN/wrn caused cellular senescence, whereas overexpression of SHOX prevented the senescence phenotype.  Figure 5d, f, it seems SHOX is not the most down-regulated gene according to the z-score and the heatmap. However, SHOX is the most significantly downregulated genes in Figure 5g, h. Can the authors explain the discrepancy?

In
Response: Thank you for your question. One possible explanation is that the three biological replicates in the control group were less than ideal, and this bias may have affected differential gene expression analysis of the WRN and control groups. However, our qPCR analysis and zebrafish FISH analysis clearly showed that loss of WRN/wrn led to downregulation of SHOX/shox. Additionally, we checked the RNA-seq dataset from Tu et al (9) and found that the SHOX gene is among the top 5 upregulated genes in WS-corrected fibroblasts, indicating that SHOX is a crucial gene regulated by WRN.  (2020) 3. In the ChiP-qRT-PCR data (Figure 8g, h), it would be better if the authors show the PCR gel images with negative and positive controls.
Response: Thank you for the suggestion. We have now added the negative and positive controls (Previous in Fig. 8g and h, now summarized in Fig. 9f and g). Additionally, we added the negative and positive controls of supple. Fig. 10a and b (now summarized in Supplementary Fig. 13e and f). All the new PCR gels results were summarized in Supplementary Fig. 13a-d in the revised manuscript. We used IgG as the negative control. We designed RNA polymerase II binding regions in human GAPDH promoter primers (192 bp) as the positive control. Anti-RNA polymerase II antibody was purchased form Thermo (26157). The results were shown below and also summarized in Supplementary. Fig. 13 a- Skeletal dysplasia and short stature are clinical phenotypes of considerable complexity with the large number of genes and spectrum of clinical disorders. SHOX is a key regulator of chondrocyte biology, as reflected by the multiple number of target genes that it regulates. While zebrafish serve as a useful model for whole body characterization of genes regulating height and skeletal components, the pathways are quite complex. Moreover, relating skeletal phenotypes of zebrafish to human and tissue-specific gene regulation is formidable. In the current work, the authors have investigated the hypothesis that the WRN helicase-nuclease implicated in the premature aging Werner syndrome regulates expression of SHOX via its G4 resolvase activity on SHOX-promoter associated G-quadruplexes.
The authors document shortened body length and bone abnormalities in wrn-/-zebrafish. From these studies they transition to human embryonic stem cells and human mesenchymal stem cells to examine the role of WRN in chondrogenesis at the cellular level. However, the direct connection of their observations remain elusive to the zebrafish model findings. To their credit, the authors assess if human WRN mRNAs injected into wrn-/-mutant zebrafish at the one-cell stage on regulation of expression of key genes in the chondrogenesis pathway. By this analysis, they find that SHOX expression is regulated by WRN during chondrocyte differentiation, and this is largely dependent on WRN helicase activity. SHOX expression on stature was further examined by microinjection of human SHOX mRNA into zebrafish embryos at the one-cell stage and embryo length was correlated with bone growth/development expression. Finally, the authors determine that WRN unwinds promoter-associated G4, thereby regulating its expression.
We truly appreciate reviewer 3 for his/her great effort reviewing our manuscript and all the valuable comments, which helped us improved the manuscript a lot.
Critical Comments: SHOX signaling is notably complex, and there is still only limited understanding of the pleiotropic effects of SHOX deficiency in humans. Therefore, the utilization of model genetic organisms with conserved genes and translational value is appreciated. Zebrafish is one such model for studying chondrogenesis, but has its limitations that are apparent at the organismal and tissue levels. A useful tool in the current study might have been G-quadruplex ligands to modulate SHOX promoter function and interrogate WRN involvement. This experimental approach might have been applied in the in vivo model.

Response:
We truly appreciate your valuable suggestions. We synthesized an oligonucleotide containing the zebrafish G4 region (z-shox-g4), as well as two oligonucleotides without z-shox-g4. We tested the formation of z-shox-G4 in vitro in a ThT assay. The fluorescent intensity (F/F0) represented the presence of G4s (a value of more than 20 is typically regarded to indicate G4 formation. Human C-MYC G4 pu18 was used as the positive control, and the pu18 mutant was used as the negative control according to previous report (10)). Fig. a (summarized in Supplementary Fig.  13j in the revised manuscript) showed that the formation z-shox-G4s in vitro, which were cloned into pGL3SV40 vectors (Promega). The dual-luciferase activity assay was conducted by transfecting z-shox-g4 or z-shox-non-g4 and full-length wrn together into sh-WRN-293T cells (the knockdown efficiency was confirmed as Fig. b shown (summarized in Supplementary Fig. 13i in the revised manuscript)). We found that full-length wrn increased shox expression more significantly compared with that in the z-shox-non-g4 groups, indicating that G4s are crucial for gene transcription in vitro (Fig. c (summarized in Fig. 9i in the revised manuscript)). However, the actual effect of these G4s in a cellular and genomic natural environment may be different. An important question is whether G4s are truly involved in transcriptional regulation in vivo during bone development. To answer this question, we disrupted G4 ligands by microinjecting z-shox anti-sense oligonucleotides (ASO) complementary to the selected G4s (Fig. d ( summarized in Fig. 9j in the revised manuscript)) into wrn -/zebrafish. We injected 10 pg-ASO/embryos or controls (an oligonucleotide that did not anneal to the zebrafish genome was used as the control (CTRL) (11)) into one-cell staged zebrafish embryos (a previous report had confirmed this optimal concentration by testing the survival of specimens until 48 hpf (11)). After confirming the efficiency of anti-sense oligonucleotides (ASO) (Fig. e (summarized in Supplementary Fig. 13k in the revised manuscript)), we performed RT-qPCR, which revealed that shox expression increased during development (Fig. f (summarized in Fig. 9k in the revised manuscript)). We also microinjected h-WRN and h-WRN helicase mutant mRNAs into wrn -/zebrafish. Overexpression of h-full-length-WRN mRNA promoted shox expression, whereas h-WRN helicase mutant did not, indicating that G4s are crucial for regulating gene expression, and WRN regulates SHOX expression by unwinding SHOX-G4s via its helicase activity. The related contents were added in the revised manuscript: Page 27 from line 679 to line 700 in results section.  Res.44. 4163-4173 (2016) Specificity of WRN's effect was not addressed. One wonders if the sequence-related BLM helicase which has also been reported to regulate gene expression via its action on G4 promoters would affect the observed phenotypes via SHOX. In my mind, it remains unclear if the effect of WRN is specific to regulation of SHOX or if other gene regulatory factors are implicated in genetic determination of height and bone homeostasis are involved, that are also directly regulated by WRN or indirectly. While the study suggests WRN is involved, a direct causal relational and mechanism is not established.
Response: Considering the reviewer's suggestion, we evaluated the BLM expression pattern during cartilage development both in vitro and in vivo. As shown below (summarized in Supplementary  Fig. 5 in the revised manuscript), there was no significant difference in the expression of BLM between CTR cells and shWRN1# and shWRN2# cell in both the hESC and hMSC models. Additionally, we measured the expression of wrn and blm at 7 dpf and 14 dpf in zebrafish; blm signals were still detected in wrn -/zebrafish, indicating that BLM/blm might not be crucial for regulating chondrogenesis and body height in WS. Ken et al suggested that functional deletion of either WRN or BLM cannot be compensated by the other protein (or other Rec Q proteins), indicating that WRN and BLM play different roles in cells (12,13). We added related contents in the revised manuscript on page 13 from line 378 to line 384 in red font in results section, and page 31 from line 783 to line 788 in red font in the discussion section. Assessment of WRN connected SHOX gene regulation on other tissues of zebrafish could be performed to provide a better impression of the broadness of the gene regulatory network. As shown by the authors, 380 genes were found to be regulated by WRN in chondrocyte homeostasis, 116 up-regulated and 264 down-regulated. Given the large number, it may be that multiple genes co-regulated with SHOX or even directly regulated by SHOX are also implicated, as suggested. It would have been informative and supportive of their model if the authors had examined in greater detail the mechanism whereby some of the top candidate involved in chondrogenesis, as evidenced by previous studies, are affected and if their regulation is relevant to the phenotypes of wrn-/-zebrafish model in this work. This would apply to SOX9, SOX5, SOX6 (The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis -PubMed (nih.gov)). The gene regulatory networks of chondrocytes are quite complex, and super enhancers as well as hostone modifications and chromatin accessibility come into play. It has been proposed that multiple transcription factors are involved in chrondrocyte biology. While the evidence from the current study suggests that WRN regulation of SHOX is among these, it is difficult to comprehend the molecular-genetic mode of action in the context of the multiplicity of transcription factors coming into play.
Response: Thank you for these suggestions and comments. We agree with the reviewer that the chondrocyte biology and regulators are complex and depends on several factors. SHOX is one of the critical factors in regulating bone development, particularly in chondrogenesis. Considering the reviewer's suggestions, we overexpressed shox by microinjecting shox at the one-cell stage and collected non-treated and treated wrn -/zebrafish at different time points. As shown below (summarized in Supplementary Fig.11), overexpression of shox promoted the expression of sox9b, sox5, and sox6 compared with that in wrn -/zebrafish. sox9 has two isoforms in zebrafish, namely sox9a and sox9b. We previously found that overexpression of shox promoted sox9a, col2a1a, and col10a1a expression in wrn -/zebrafish. (data please refers to Fig. 7k-p and Fig. 7r-w)). Taken together, these data suggested that shox co-regulated with sox9a, sox9b, sox5, and sox6 in chondrogenesis. Our study is agreed with previous report that SHOX can physically interact with various proteins, such as SOX9, SOX5, and SOX6, to promote bone development (14). Additionally, Mirial et al reported that SHOX directly interacts with SOX5 and SOX6 and that mutations in SHOX inhibite the interactions (15). Moreover, SHOX, SOX9, SOX5, and SOX6 were coexpressed in 18and 32-week human fetal growth plates, indicating that SHOX coregulate these genes during skeletal development and determination of the body stature (15). However, whether SHOX can directly interact with SOX9 is unclear. Shox can co-regulate with Sox9 (Sox9 plays a crucial role in chondrogenesis by promoting the expression of cartilage matrix genes, such as Col2a1a and Aggrecan, which may further promote skeletal growth and development) in promoting chicken limb growth (16). We added related contents in the revised manuscript on page 22 from line 564 to line 569 in red font in results section, and page 33 from line 836 to line 840 in red font in the discussion part.  Endocr Rev.37, 417-448 (2016) Based on their results, the authors suggest that WRN controls bone growth and development via its regulation of SHOX. Are there other phenotypes beyond bone-related affected by WRN status and are these also mediated by SHOX? The complexity of WRN's genetic linkage to range of clinical features of accelerated aging. This is compounded by the extensive data that at least in vitro (biochemical), cell-based models, and genetic models, WRN plays roles in transcriptional regulation, DNA repair, replication stress response, genomic stability. So is this an oversimplification that WRN's involvement in controlling bone metabolism in zebrafish is mediated by its effect on SHOX, and there is no consideration of its pan-wide functions in multiple cell types and tissues?
Response: Thank you for raising this important point. Considering the reviewer's question, qRT-PCR was performed to evaluate the expression patterns of wrn and shox at 40 dpf both in wildtype and wrn -/zebrafish (most zebrafish tissues and organs have formed at this stage). As shown below (also summarized in Supplementary Fig.7g-h in the revised manuscript), the wrn gene was universally expressed in the wildtype zebrafish, and the shox gene mainly expressed in the bones (vertebrate bone and craniofacial bone) and slightly in the brain, indicating that shox mainly participates in bone tissue related activities, in agreement with a previous report (17) Another layer of potential complexity is added with the idea that WRN's key molecular activity of G4 resolution in the promoter of SHOX underlies the growth/bone phenotypes may be overly simplified. WRN, along with other G4-resolving helicases (e.g., BLM) is thought to play a role in regulation of expression of many genes with G4-laden promoters, not to mention its effect on G4 in other chromosomal regions (e.g., telomeres). I'm not sure all the phenotypes related to chrondrocyte biology hinge on WRN-catalyzed G4 resolution of SHOX promoter DNA sequence elements. This seems to be the driving conclusion of the paper, and I am not sure that other avenues have been explored which potentially contribute to the observations made for wrn-/-zebrafish.
Response: Thank you for raising the important point. It has previously been reported that WRN preferentially binds to a "bubble" structure, such as duplex DNA and G4 quadruplex, where it functions as a helicase to open these structures to promote DNA transcription, duplication, and replication (18). Gene promoters correlate highly with gene expression. As described in our response to Question 1, we synthesized a zebrafish G4 region which was cloned into the pGL3SV40 vector. We co-transfected z-shox-g4-pGL3SV40 with the zebrafish full-length wrn plasmid into shWRN-293T cells. These cells showed increased shox expression compared with that in the control cells ( Fig. a-b, summarized in Supplementary Fig. 13j and Fig. 9i). To examine whether WRN preferentially resolves G4 structures, we synthesized two regions in the promoter without zebrafish G4 quadruplexes and performed the dual luciferase assays again. As shown below (Fig. a-b, summarized in Supplementary Fig. 13j and Fig. 9i), we found that wrn could promote shox expression significantly especially if there was a G4 structure in the promoter, indicating that WRN helicase is more sensitive to recognize a G4 structure and open it for further gene transcription and expression. X-ray crystallographic analysis has shown that WRN helicase preferred to bind to these "bubble structures" due to its unique winged-helix domain of WRN protein (19). In the meantime, we noted that the disruption of z-shox-g4 ligands enhanced chondrogenesis, similar effect was observed when microinjecting h-full-length-WRN mRNAs, indicating that WRN-catalyzed SHOX-G4-promoter is crucial in regulating chondrogenesis (Fig. c-d, summarized in Fig. 9l-n).
We added related contents in the revised manuscript on page 27 from line 679 to line 704 in red font in results section, and page 33 from line 857 to line 865 in discussion section.  (2021) Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes; Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes -PubMed (nih.gov) reported that wrn-/-zebrafish. Like mre11-/-zebrafish and certain other DNA repair mutants displayed reduced growth and development during the juvenile stage. Interestingly, both mre11acu56/cu56 and wrncu64/cu64 failed to survive to 60 dpf. The similar phenotype raises the question if the genetic defect is attributed to unusual G4 metabolism or more generically a DNA repair defect such as double-strand break repair in which both WRN and MRE11 are implicated. It would have been of interest if the authors of the current study had determined what phenotypes and at what developmental stage were observed in wrn-/-zebrafish that had been exposed to a G4binding ligand. If the temporal appearance or severity of the observed phenotypes were G4-inducible, then this would support their model.
Response: We truly appreciate your valuable comments. Unusual G4 metabolism is associated with DNA repair defects, DNA damage etc (20). We examined the expression of γH2AX in both wildtype and wrn -/zebrafish. As shown in Fig. a below (also summarized in Supplementary Fig. 2b in the revised manuscript), the expression of γH2AX in the vertebrate regions increased in wrn -/zebrafish as developmental stages proceed. As described in our response to Question 1, we designed an antisense oligonucleotide (ASO) targeting zebrafish shox-G4 ligands and microinjected this ASO at the one-cell stage (Fig. b, summarized in Fig. 9j in the revised manuscript). After confirming the efficiency of shox-g4-ASOs (Fig. c, summarized in Supplementary Fig. 13k in the revised manuscript), we evaluated shox expression at different developmental stages, which was found to be increasing over time compared with that in the control groups (Fig. d, summarized in Fig. 9k in the revised manuscript). Additionally, we microinjected the human full-length WRN mRNA and the human helicase mutant mRNA respectively (Fig. d, summarized in Fig. 9k in the revised manuscript). Compared to ASO group, overexpression of human WRN mRNA in wrn -/zebrafish increased shox gene expression, whereas the human helicase mutant did not. Together, these data demonstrate that G4 structures are present at an early developmental stage, and that their formation is inducible. We added related contents in the revised manuscript on page 27 from line 689 to line 704 in red font in results section. The Short-Stature Homeobox-Containing Gene ( shox/ SHOX) Is Required for the Regulation of Cell Proliferation and Bone Differentiation in Zebrafish Embryo and Human Mesenchymal Stem Cells -PubMed (nih.gov)) had assessed the phenotypes of morpholino oligo-mediated knockdown of zebrafish shox. Determination if wrn and shox genetically operate in the same pathway as predicted by the model proposed by the authors of the current study would have helped to strengthen the study.
Response: Thank you for your comments. As we previously discussed, the expression pattern of WRN and SHOX were highly similar during chondrogenesis in both hESCs and hMSCs (Fig. a-d).
Since chondrogenesis initiates from mesendoderm and the cells undergo mesenchymal condensation, proliferation and a serious of other delicate events, they finally become chondrocytes (5). Our results revealed that WRN and SHOX proteins both appeared from the primitivemesendoderm population (day 4, Fig. a-b) to the mesoderm population (day 9, Fig. a-b), and were also detected in chondrocytes (day 14, Fig. a-b). (Fig. a is summarized in Fig. 3a, Fig. b summarized in Supplementary Fig. 8a). Similar results were observed in the hMSC model (Fig. c (summarized in Supplementary Fig. 3b), Fig. d (summarized in Supplementary Fig. 9a)). These data indicate that both WRN and SHOX participate in the very early stage of chondrogenesis, as confirmed by immunostaining (Fig. e (summarized in Fig. 6a), Fig. f. (summarized in Supplementary Fig.9c)). Similar to the results observed in cells, wrn and shox were both expressed early from 3 to 14 dpf along the vertebrate regions in wildtype zebrafish but was not in wrn -/mutant zebrafish ( Fig. g- Supplementary Fig.7h-j). Additionally, loss of WRN or SHOX may lead to decreased expression of Ki67, a proliferative marker, indicating that both genes are involved in proliferation ( Fig. j (summarized in Fig. 3u RecQ Helicases (nih.gov) might be relevant to the current work, although in that paper Blm and Recql5 were the focus. Many thanks for sharing this interesting work with us, and it is helpful for our study.
Overall Critique: The study of Tian et al. invokes a model in which zebrafish WRN has importance for bone development and growth, a finding consistent with another recently published study in DNA repair. The apparent advance here is that short stature homobox gene SHOX expression is regulated by WRN, which may be a causative factor for wrn-/-related phenotypes; however, further experimental studies might have more strongly supported this conclusion. Furthermore, the authors of the current study propose that WRN regulates SHOX gene expression via resolution of promoter Gquadruplexes of the SHOX gene; however, it is unclear how specific this function of WRN truly is, given that there are a number of G4-resolving helicases in humans. Extrapolation of the findings from zebrafish to human remains uncertain and likely would be more complex in the higher order vertebrate. It remains to be fully understood if the dysregulation of SHOX due to loss of WRN G4 resolvase activity underlies the short stature in humans with WS, or if the molecular-genetic basis is more complex.